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. 2010 Oct 4:10:526.
doi: 10.1186/1471-2407-10-526.

Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

Francesco Piazza  1 , Sabrina Manni, Laura Quotti Tubi, Barbara Montini, Laura Pavan, Anna Colpo, Marianna Gnoato, Anna Cabrelle, Fausto Adami, Renato Zambello, Livio Trentin, Carmela Gurrieri, Gianpietro Semenzato
Affiliations

Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

Francesco Piazza et al. BMC Cancer. .

Abstract

Background: Glycogen Synthase Kinase-3 (GSK-3) α and β are two serine-threonine kinases controlling insulin, Wnt/β-catenin, NF-κB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3α and GSK-3β function in multiple myeloma (MM).

Methods: GSK-3 α and β expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 α and β isoforms. Survival signaling pathways were studied with WB analysis.

Results: GSK-3α and GSK-3β were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3β knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of β-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself.

Conclusions: These data suggest that in MM cells GSK-3α and β i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors.

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Figures

Figure 1
Figure 1
GSK-3 expression, phosphorylation and localization in MM cells. (A) WB for total GSK-3α and β (top panels), phosphorylated GSK-3α (on Ser 21) and GSK-3β (on Ser 9) (middle panels), and β-actin as loading control (bottom panels) in (from left to right): 4 PBMC samples of healthy subjects, non-malignant in vitro-generated PC (nPC), CD138+ purified PC from 9 MM patients, 4 MMCLs; (B) WB for total GSK-3α and β (top panels), phosphorylated GSK-3α (on Tyr 279) and GSK-3β (on Tyr 216) (middle panels) and β-actin as loading control (bottom panels), in three primary MM plasma cell samples (MM 7, 3, 1). (C-E): GSK-3 immunostaining and confocal microscopy analysis in MMCLs OPM-2, INA-6, U-266 and RPMI-8226 (C), three CD138+ primary malignant PC from patients (MM1, MM9, MM10) (D) and BMMC (E). In the merged images GSK-3 is detected by green fluorescence and nuclei are in blue. Scale bars = 10 μm. For all the images: 600× magnification, oil objective.
Figure 2
Figure 2
Effects of GSK-3 inhibitors on MM cell proliferation. (A, B) Graphs showing representative in vitro kinase assays performed using human recombinant GSK-3β (hrGSK-3β) and total protein lysates (+ = 1 μg, ++ = 4 μg) of OPM-2 cells (A) and RPMI-8226 cells (B) that were cultured with the two specific GSK-3 inhibitors, SB216763 at 10 μM and SB415286 at 4 μM. The graphs show a remarkable inhibition of both hrGSK-3β protein (leftmost graphs) and endogenous GSK-3 kinase activity (rightmost graphs) (y axis: counts per minute). Data represent mean ± SD, n = 3. * indicates p < 0.05 by one-way ANOVA test. (C) Dose-response graphs of U-266 (left) and INA-6 cells (right) incubated for 48 hours with increasing concentrations of SB415286. Cell proliferation was assessed by 3H-thymidine-incorporation assay. Data represent mean ± SD, n = 4, * indicates p < 0.05 by one-way ANOVA test.
Figure 3
Figure 3
GSK-3 inhibition causes MM cell apoptosis and disruption of the mitochondrial membrane potential. (A) Histograms showing the percentage of surviving (annexin V negative) U-266, RPMI-8226 and INA-6 cells (left graph) and of normal PBMC control cells (right graph) after treatment without (black bars) or with (white bars) 4 μM SB415286 for 48 hours. Data represent mean ± SD, n = 5 (cell lines) or 4 (PBMC). * indicates p < 0.05 by Student's t-test. (B) Histograms showing FACS analysis of the amount of JC-1 monomer-containing, green fluorescent-INA-6 cells (left graph) or U-266 cells (right graph), treated with the same conditions as in A. Data are expressed as ratio over untreated cells. Data represent mean ± SEM, n = 5. * indicates p < 0.01 by Student's t-test. (C) Representative WB analysis of phospho Tyr 279 GSK-3α and phospho Tyr 216 GSK-3β, total GSK-3, PARP cleavage and Smac/DIABLO protein expression on cell lysates of RPMI-8226 cells grown with 5 μM or 10 μM SB216763 for 24 hours (top panels) and WB analysis of PARP protein in INA-6 cells grown with 4 μM SB415286 for 8 and 24 hours (bottom). β-Actin is used to check protein loading. (D) Representative WB analysis of nuclear (n) and cytosolic (c) proteins from U-266 cells untreated or treated with 5 μM SB216763, showing the levels of total GSK-3, Tyr phosphorylated GSK-3, β-catenin, total ERK 1/2, Thr/Tyr phosphorylated ERK 1/2, PARP and nucleophosmin and GAPDH as loading controls.
Figure 4
Figure 4
GSK-3 inhibition increases MM cell sensitivity to bortezomib-induced apoptosis. (A) Histograms summarizing the data on the percentage of apoptotic MM cells, evaluated by annexin V staining and FACS analysis, left untreated (white bars) treated with the GSK-3 inhibitor SB415286 (4 μM) (black bars), bortezomib (BZ) 5 nM (black and white dashed bars) or both (black, white-dotted bars). Top graphs: U-266 cells, bottom: RPMI-8226 cells. Data represent mean ± SEM, n = 3. * indicates p < 0.05 by one-way ANOVA test. (B) Representative WB analysis of nuclear (n) and cytosolic (c) proteins from U-266 cells untreated, treated with 4 μM SB415286, with 5 nM BZ or both, showing the levels of PARP and cleaved PARP, Ser 473-phosphorylated AKT, AKT and MCL-1. Nucleophosmin and GAPDH were used as nuclear and cytosolic loading controls, respectively.
Figure 5
Figure 5
Effects of GSK-3α and β knockdown by RNA interference on MM cell survival. (A) WB analysis of the levels of GSK-3α (left panels) or GSK-3β (right panels) upon nucleofection with: un = untransfected; scr = scrambled siRNAs oligos; GSK-3α or GSK-3β=GSK-3α or GSK-3β-directed siRNAs oligos. (B, left panels) Light microscopy microphotographs of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos. Scale bars = 10 μm. (B, middle plots) Representative dot plot graphs and FACS analysis of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos. (B, right panels) Histograms summarizing the results of FACS analysis of U-266 cells transfected with scrambled (scr) siRNAs oligos, GSK-3α (top) or GSK-3β (bottom)-specific siRNAs oligos,. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 6. * indicates p < 0.05 (Student's t test). (C) Representative WB analysis of the levels of unprocessed PARP after nucleofection of U-266 cells with: scrambled, GSK-3α or GSK-3β siRNAs oligos. (D) Light microscopy microphotographs and histograms summarizing the results of FACS analysis of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos and treated with 5 nM BZ. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 3. * indicates p < 0.05 (one-way ANOVA test). (E) Histogram plot summarizing the results of the FACS analysis of U-266 cells transfected with scrambled (scr) or GSK-3α/GSK-3β-directed siRNA oligos and treated with 5 nM BZ. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 3. * indicates p < 0.05 (one-way ANOVA). (F) WB analysis of GSK-3α, GSK-3β, AKT, Ser 473-phosphorylated AKT and MCL-1 protein levels in U-266 cells untransfected (lane 1), treated with 5 nM BZ (lane2), transfected with scrambled (scr, lane 3) or GSK-3α/β (lane 4), GSK-3α (lane 5) or GSK-3β (lane 6)-directed siRNAs oligos without (-, lanes 3-6) or with (+, lanes 7-10) exposure to 5 nM BZ for 18 hours.
Figure 6
Figure 6
Bortezomib modulates GSK-3 intracellular localization and activation. (A) Immunofluorescence microscopy of INA-6 and U-266 cells untreated or treated with 5 nM BZ for 18 hours and stained for GSK-3 (green fluorescence) and the nuclei (DAPI, blue fluorescence) showing partial nuclear re-localization of GSK-3 upon BZ treatment. Scale bars = 10 μm. On the bottom, graph showing the percentage of MM cells scored as having cytosolic only GSK-3 in the untreated (black bars) or 5 nM BZ-treated (white bars) conditions. Data represent mean ± SD, n = 3. * indicates p < 0.05. (B) Representative WB analysis of phospho Ser 9 GSK-3β/Ser 21 GSK-3α and total GSK-3α/β in nuclear (n) and cytoplasmic (c) protein fractions from untreated (-) or 5 nM BZ-treated (18 hours) U-266 cells. β-Actin and nuclear PARP are used as markers for protein loading. (C) Representative WB analysis of phospho Tyr 279 GSK-3α/Tyr 216 GSK-3β and total GSK-3α/β in nuclear (n) and cytoplasmic (c) protein fractions from untreated (-) or 5 nM BZ-treated (+) (18 hours) U-266 cells, in the presence or absence of 5 μM or 10 μM SB216763. GAPDH and nucleophosmin were used as protein loading markers.
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